Name: biophysical characterization of flagellar motor functions
Uploaded: 2017-01-18T15:00:00
Description: Watch this Scientific Journal Video about Biophysical Characterization of Flagellar Motor Functions at JoVE.com

The authors acknowledge Howard Berg for the gift of the bead-tracking microscope/photomultipliers and the Texas A&amp;M Engineering Experiment Station for funds.

1. Cell Preparation
<ol>
	<li>Grow overnight cultures of the desired strain carrying the sticky fliC allele 15,22 in Tryptone Broth (TB, 1% Peptone, 0.5% NaCl) followed by inoculation at 1:100 dilution in 10 mL fresh TB. Grow the culture at 33 &#176;C in a shaker incubator until OD600 = 0.5.</li>
	<li>Pellet the cells at 1,500 x g for 5 - 7 min and re-disperse the pellet vigorously in 10 mL of filter sterilized motility buffer (MB; 10 mM phosphate buffer: 0.05-0.06 M NaCl, 10-4...

<ol>
	<li>Emody, L., Kerenyi, M., Nagy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+factors+of+uropathogenic+Echerichia+coli.">Virulence factors of uropathogenic Echerichia coli.</a> Int J Antimicrob. Ag. 22, 29-33 (2003).</li><li>Lane, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+motility+in+the+colonization+of+uropathogenic+Escherichia+coli+in+the+urinary+tract.">Role of motility in the colonization of uropathogenic Escherichia coli in the urinary tract.</a> Infect Immun. 73 (11), 7644-7656 (2005).</li><li>Kao, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+interplay+among+bacterial+motility+and+virulence+factors+in+different+Escherichia+coli+infections.">The complex interplay among bacterial motility and virulence factors in different Escherichia coli infections.</a> Eur J Clin Microbiol Infect Dis. 33 (12), 2157-2162 (2014).</li><li>Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rotary+motor+of+bacterial+flagella.">The rotary motor of bacterial flagella.</a> Annu Rev Biochem. 72, 19-54 (2003).</li><li>McCarter, L., Hilmen, M., Silverman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellar+Dynamometer+Controls+Swarmer+Cell+Differentiation+of+V.+parahaemolyticus.">Flagellar Dynamometer Controls Swarmer Cell Differentiation of V. parahaemolyticus.</a> Cell. 54 (3), 345-351 (1988).</li><li>Lele, P. P., Hosu, B. G., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+mechanosensing+in+the+bacterial+flagellar+motor.">Dynamics of mechanosensing in the bacterial flagellar motor.</a> Proc Natl Acad Sci U S A. 110 (29), 11839-11844 (2013).</li><li>Gode-Potratz, C. J., Kustusch, R. J., Breheny, P. J., Weiss, D. S., McCarter, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+sensing+in+Vibrio+parahaemolyticus+triggers+a+programme+of+gene+expression+that+promotes+colonization+and+virulence.">Surface sensing in Vibrio parahaemolyticus triggers a programme of gene expression that promotes colonization and virulence.</a> Mol Microbiol. 79 (1), 240-263 (2011).</li><li>Kearns, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+field+guide+to+bacterial+swarming+motility.">A field guide to bacterial swarming motility.</a> Nat Rev Microbiol. 8 (9), 634-644 (2010).</li><li>Belas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms,+flagella,+and+mechanosensing+of+surfaces+by+bacteria.">Biofilms, flagella, and mechanosensing of surfaces by bacteria.</a> Trends Microbiol. 22 (9), 517-527 (2014).</li><li>Silverman, M., Simon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellar+rotation+and+the+mechanism+of+bacterial+motility.">Flagellar rotation and the mechanism of bacterial motility.</a> Nature. 249, 73-74 (1974).</li><li>Block, S. M., Segall, J. E., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+Kinetics+in+Bacterial+Chemotaxis.">Adaptation Kinetics in Bacterial Chemotaxis.</a> J Bacteriol. 154 (1), 312-323 (1983).</li><li>Segall, J. E., Block, S. M., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+comparisons+in+bacterial+chemotaxis.">Temporal comparisons in bacterial chemotaxis.</a> Proc Natl Acad Sci U S A. 83, 8987-8991 (1986).</li><li>Blair, D. F., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+torque+in+defective+flagellar+motors.">Restoration of torque in defective flagellar motors.</a> Science. 242 (4886), 1678-1681 (1988).</li><li>Ryu, W. S., Berry, R. M., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torque-generating+units+of+the+flagellar+motor+of+Escherchia+coli+have+a+high+duty+ratio.">Torque-generating units of the flagellar motor of Escherchia coli have a high duty ratio.</a> Nature. 403, 444-447 (2000).</li><li>Yuan, J., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resurrection+of+the+flagellar+rotary+motor+near+zero+load.">Resurrection of the flagellar rotary motor near zero load.</a> Proc Natl Acad Sci U S A. 105 (4), 1182-1185 (2008).</li><li>Yuan, J., Fahrner, K. A., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switching+of+the+bacterial+flagellar+motor+near+zero+load.">Switching of the bacterial flagellar motor near zero load.</a> J Mol Biol. 390 (3), 394-400 (2009).</li><li>Sowa, Y., Hotta, H., Homma, M., Ishijima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torque-speed+Relationship+of+the+Na+-driven+Flagellar+Motor+of+Vibrio+alginolyticus.">Torque-speed Relationship of the Na+-driven Flagellar Motor of Vibrio alginolyticus.</a> J Mol Biol. 327 (5), 1043-1051 (2003).</li><li>Xing, J., Bai, F., Berry, R., Oster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torque-speed+relationship+of+the+bacterial+flagellar+motor.">Torque-speed relationship of the bacterial flagellar motor.</a> Proc Natl Acad Sci U S A. 103 (5), 1260-1265 (2006).</li><li>Meacci, G., Tu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+bacterial+flagellar+motor+with+multiple+stators.">Dynamics of the bacterial flagellar motor with multiple stators.</a> Proc Natl Acad Sci U S A. 106 (10), 3746-3751 (2009).</li><li>Lele, P. P., Roland, T., Shrivastava, A., Chen, Y. H., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+flagellar+motor+of+Caulobacter+crescentus+generates+more+torque+when+a+cell+swims+backwards.">The flagellar motor of Caulobacter crescentus generates more torque when a cell swims backwards.</a> Nat Phys. 12 (2), 175-178 (2016).</li><li>Lele, P. P., Shrivastava, A., Roland, T., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+thresholds+in+bacterial+chemotaxis.">Response thresholds in bacterial chemotaxis.</a> Sci Adv. 1 (9), e1500299 (2015).</li><li>Berg, H. C., Turner, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torque+Generated+by+the+Flagellar+Motor+of+Escherichia+coli.">Torque Generated by the Flagellar Motor of Escherichia coli.</a> Biophys J. 65, 2201-2216 (1993).</li><li>Bai, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Spread+as+a+Mechanism+for+Cooperativity+in+the+Bacterial+Flagellar+Switch.">Conformational Spread as a Mechanism for Cooperativity in the Bacterial Flagellar Switch.</a> Science. 327, 685-689 (2010).</li><li>Reid, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maximum+number+of+torque-generating+units+in+the+flagellar+motor+of+Escherichia+coli+is+at+least+11.">The maximum number of torque-generating units in the flagellar motor of Escherichia coli is at least 11.</a> Proc Natl Acad Sci U S A. 103, 8066-8071 (2006).</li><li>Chen, X., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torque-Speed+Relationship+of+the+Flagellar+Rotary+Motor+of+Escherichia+coli.">Torque-Speed Relationship of the Flagellar Rotary Motor of Escherichia coli.</a> Biophys J. 78, 1036-1041 (2000).</li><li>Turner, L., Caplan, S. R., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-induced+switching+of+the+bacterial+flagellar+motor.">Temperature-induced switching of the bacterial flagellar motor.</a> Biophys J. 71, 2227-2233 (1996).</li></ol>

In order to facilitate tethered bead-tracking and correct estimation of motor-torques, the following information should be reviewed. When performing these measurements with flagellated cells, shearing is a critical step. Shearing reduces the flagellar filament to a mere stub, thereby ensuring that the viscous load on the motor is predominantly due to the bead and can be estimated within 10% error 16. Shearing also improves the chances of finding circular trajectories with tightly distributed eccentricities (&#...

Flagellar motors enable cells to swim by rotating helical extracellular filaments. The amount of torque the motor can generate for a given length of the flagellum (i.e., the viscous load) determines the swimming speeds. On the other hand, its ability to switch the direction of rotation controls cell migration in response to chemicals, a process known as chemotaxis. Chemotaxis and motility being virulence factors 1-3, flagellar motors have been well-characterized over the years 4. Mounting evidence now suggests that the motor acts as a mechanosensor &#8212; it mechanically detects the presence of solid substrates 5,6. This ability likely helps in triggering surface colonization and infections 5,7. As a result, the mechanisms whereby the motor senses surfaces and initiates signaling are of significance 8,9.
The flagellar motor can be readily studied by tethering the flagellum to a substrate and observing cell rotation. Such tethering was first achieved by Silverman and Simon, who worked with a polyhook mutant in E. coli and successfully attached hooks to glass substrates with anti-hook antibodies 10. The tethered-cell assay enabled researchers to study the responses of the motor-switch to a variety of chemical stimuli. For example, Segall and co-workers chemically stimulated tethered cells with the aid of iontophoretic pipettes. The corresponding changes in CWbias (the fraction of the time motors spin clockwise, CW) enabled them to measure the kinetics of adaptation in the chemotaxis network 11,12. While the tethered cell assay was effective in studying switch responses, it was only able to offer insights into motor mechanics over a limited range of viscous loads 13. To overcome this problem, Ryu and co-workers tethered spherical, latex beads to filament stubs on cells stuck to surfaces. The beads were then tracked using back-focal interferometry with weak optical traps 14. By working with beads of different sizes, researchers could study the motor over a much wider range of loads. This assay was later improved by Yuan and Berg, who developed a photomultiplier-based bead-tracking technique combined with laser dark-field illumination. Their method enabled tracking of tethered gold nanobeads that were so tiny (~ 60 nm) that the external viscous resistances were lower compared to the internal viscous resistances to rotation 15,16. This led to the measurements of the maximum achievable speeds in E. coli (~ 300 Hz). In V. alginolyticus, similar bead assays enabled measurements of the spinning rates at intermediate viscous loads (~ 700 Hz) 17. By enabling measurements of motor responses over the entire possible range of viscous loads (from zero-load to near-stall), the bead-assays provided an important biophysical tool to understand the torque-generation process 18,19.
Recently, we modified the Yuan-Berg assay to include optical tweezers that enabled us to apply precise mechanical stimuli to individual motors 6. Using this technique, we showed that the force-generators that rotate the motor are dynamic mechanosensors &#8212; they remodel in response to changes in viscous loads. It is possible that such load-sensing triggers cell differentiation into swarming bacteria, although the mechanisms remain unclear. It is also likely that the flagellar motors in other species are also mechanosensitive 20, although direct evidence is lacking. Here, we discuss the photomultiplier-based (PMT) approach for tracking the rotation of latex beads tethered to flagellar filaments 15. In comparison to tracking with ultrafast cameras, the photomultiplier-setup is advantageous because it is relatively straightforward to track single beads in real-time and over long durations. It is particularly useful when studying long-time remodeling in flagellar motor complexes due to environmental stimuli 21. Though we detail protocols specifically for E. coli, they can be readily adapted for studying flagellar motors in other species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Poly-L-lysine Solution (0.1%)</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en&#38;region=US</td>
		</tr>
		<tr>
			<td>Polybead Microspheres</td>
			<td>Polysciences, Inc.</td>
			<td>7307</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en&#38;region=US</td>
		</tr>
		<tr>
			<td>1 ml Luer Slip Tip Syringe</td>
			<td>Exel Int.</td>
			<td>26048</td>
			<td>http://www.exelint.com/tuberculin_syringes.php</td>
		</tr>
		<tr>
			<td>Clay Adams Intramedic Luer-Stub Adapter 23-gauge</td>
			<td>Becton, Dickinson and Company</td>
			<td>427565</td>
			<td>http://www.bd.com/ds/productCenter/ES-LuerStubAdaptors.asp</td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Harvard Apparatus</td>
			<td>59-8325</td>
			<td>http://www.harvardapparatus.com/laboratory-polye-polyethylene-non-sterile-tubing.html</td>
		</tr>
		<tr>
			<td>Photomultiplier Tubes</td>
			<td>Hamamatsu</td>
			<td>R7400U-20</td>
			<td>Spectral response range of 300 to 920 nm, Peak wavelength 630 nm,&#160; 0.78 ns response time&#160; 
			http://pdf1.alldatasheet.com/datasheet-pdf/view/212308/HAMAMATSU/R7400U-20.html</td>
		</tr>
		<tr>
			<td>3x1 mm precision slits</td>
			<td>Edmund Optics</td>
			<td>NT39-908</td>
			<td>2 slits mounted at right angles to one another on photomultiplier tubes</td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix</td>
			<td>TBS 1032B</td>
			<td>Alternative brands are acceptable. Digital Oscilloscope, TBS 1000B Series, 2 Analogue, 30 MHz, 500 MSPS, 2.5 kpts&#160; 
			http://www.tek.com/oscilloscope/tbs1000b-digital-storage-oscilloscope</td>
		</tr>
		<tr>
			<td>8 Pole LP/HP Filter</td>
			<td>Krohn-Hite</td>
			<td>3384</td>
			<td>Alternative brands are acceptable. A frequency range from 0.1 Hz to 200 kHz is recommended.&#160;&#160;&#160; 
			http://www.krohn-hite.com/htm/filters/PDF/3384Data.pdf</td>
		</tr>
		<tr>
			<td>Optiphot microscope</td>
			<td>Nikon</td>
			<td>NA</td>
			<td>Any upright or inverted phase microscope can be used. 
			https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=754</td>
		</tr>
		<tr>
			<td>50:50 (R:T) Cube Beamsplitter</td>
			<td>ThorLabs</td>
			<td>BS013</td>
			<td></td>
		</tr>
	</tbody>
</table>

biophysical characterization of flagellar motor functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

The photomultiplier setup is shown in Figure 1A. It is important that the PMTs have high sensitivities over the range of wavelengths scattered by the beads of interest. The PMTs employed here operate in the visible and near-infrared ranges, and were able to detect light scattered by beads illuminated by a halogen light source. The optimum lighting conditions and supply voltages will vary from one setup to another. For the setup used in this work, a PMT gain ~ 104</su...

Watch this Scientific Journal Video about Biophysical Characterization of Flagellar Motor Functions at JoVE.com

Biophysical Characterization of Flagellar Motor Functions

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...

The overall of goal of this experimental procedure is to measure the rotation of probe beads attached to individual flagellar motors in live bacterial cells. The technique that we'll describe offers the advantage of real-time tracking of motor responses and the ability to discriminate between admissible and inadmissible data in real time. The approach is helping us answer key questions in the field of mechanobiology and uncover the physics underlying molecular motor behavior.Although we predominantly employ these techniques to study flagellar motors in E.coli, these protocols can be readily adapted to study motors in other species of flagellated bacteria. Grow overnight cultures of the desired strain carrying the sticky fliC allele in TB.The next day, inoculate 10 milliliters of fresh TB at a 1:100 dilution. Grow the culture at 33 degrees Celsius in a shaker incubator until the optical density at 600 nanometers equals 0.5.Pellet the cells at 1500 times G for five to seven minutes. Then, redisperse the pellet vigorously in 10 milliliters of filter-sterilized motility buffer. Repeat the centrifugation and resuspension two more times.Redisperse the final pellet in one milliliter of MB.Shear the suspension by passing it back and forth approximately 75 times between two syringes with 21 to 23 gauge adapters connected by polyethylene tubing. Limit the total time for shearing to 30 to 45 seconds. After centrifuging the sheared cells, redisperse the pellet in 100 to 500 microliters of MB.Prepare an imaging chamber by sandwiching two double-sided adhesive tapes between a cover slip and a microscope slide.Add 0.01%poly-L-lysine solution in the chamber and after five minutes, gently rinse the surfaces with MB.Add 40 microliters of the cell suspension into the chamber and allow sufficient time for attachment to the glass surface. Flow out unstuck cells by adding 100 microliters of MB on one side of the chamber, while wicking the solution with filter paper from the other side. Next, add 10 to 15 microliters of latex beads into the chamber.Use a range of bead sizes for the experiments that result in a good contrast. Allow the beads adequate time to settle and attach to the cells. Gently rinse the beads with 100 microliters of MB to remove unstuck beads.Place the sample on a microscope stage and scan the surface for beads attached to motors. Employ bright-field imaging with a 60X objective as long as sufficient contrast is maintained to clearly distinguish a bright bead on a dark background. Once a bead has been selected, move the stage laterally to position the bead in a pre-determined corner.Position beads at the same corner to ensure that the direction of rotation of the bead is correctly known. Maintain the sampling frequency higher than twice the rotational frequency of the motor to avoid errors associated with aliasing. In this work, sampling frequencies that were 10 times higher were used to observe a motor rotating at 50 hertz.If, after tethering, the beads appear to wobble with an eccentricity greater than one to two times that of the bead radius, then it's recommended to shear the cells a greater number of times. Shown here is a visual comparison between an appropriately-tethered bead and a bead that is not tethered. The outputs from the two photomultipliers, sampled at 500 hertz, are represented over a small duration of the measurement.The filtered outputs from the PMTs, after centering and scaling, were used to reconstruct the circular trajectories of the rotating bead, as shown here. The ideal bead trajectory is approximately circular but elliptical trajectories are admissible. The angular speeds calculated from the bead trajectories for a single motor are represented over a three-second window.The repeated transitions of the motor between the counterclockwise and clockwise directions of rotation are indicated by switches between positive and negative values. After watching this video, you should have a good understanding of how to track beads tethered to single flagellar motors with high accuracy, using the photomultiplier-based techniques presented here. The bacterial flagellar motor presents a unique set of challenges because it is capable of rotating at speeds of several hundred hertz.High accuracy tracking of probe beads tethered to molecular motors facilitated the characterization of several types of motors, including kinesins, myosins and F1-ATPase. The extension of the bead tethering technique to flagellar motors enabled researchers to study motor mechanics with an unprecedented accuracy. Visual demonstration of this method will help researchers wishing to utilize this technique to overcome challenges such as locating beads appropriately tethered to individual motors.It is expected that the methods demonstrated here will enable the development of novel insights into the adaptability of these nanomachines in a variety of bacterial species.

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Biology

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 &#176;C instead of the conventional 70-100 &#176;C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other &#39;large&#39; proteins like BRCA2 should be readily achievable.

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...